Suppression of prion protein in livestockby RNA interferenceMichael C. Golding*, Charles R. Long†, Michelle A. Carmell*, Gregory J. Hannon*‡, and Mark E. Westhusin†§
*Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Howard Hughes Medical Institute, 1 Bungtown Road, Cold Spring Harbor, NY 11724;and †Department of Veterinary Physiology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843
Communicated by James E. Womack, Texas A&M University, College Station, TX, February 2, 2006 (received for review November 21, 2005)
Given the difficulty of applying gene knockout technology tospecies other than mice, we decided to explore the utility of RNAinterference (RNAi) in silencing the expression of genes in live-stock. Short hairpin RNAs (shRNAs) were designed and screened fortheir ability to suppress the expression of caprine and bovine prionprotein (PrP). Lentiviral vectors were used to deliver a transgeneexpressing GFP and an shRNA targeting PrP into goat fibroblasts.These cells were then used for nuclear transplantation to producea cloned goat fetus, which was surgically recovered at 81 days ofgestation and compared with an age-matched control derived bynatural mating. All tissues examined in the cloned fetus expressedGFP, and PCR analysis confirmed the presence of the transgeneencoding the PrP shRNA. Most relevant, Western blot analysisperformed on brain tissues comparing the transgenic fetus withcontrol demonstrated a significant (>90%) decrease in PrP expres-sion levels. To confirm that similar methodologies could be appliedto the bovine, recombinant virus was injected into the perivitellinespace of bovine ova. After in vitro fertilization and culture, 76% ofthe blastocysts exhibited GFP expression, indicative that theyexpressed shRNAs targeting PrP. Our results provide strong evi-dence that the approach described here will be useful in producingtransgenic livestock conferring potential disease resistance andprovide an effective strategy for suppressing gene expression in avariety of large-animal models.
nuclear transfer � short hairpin RNA � transgenic � bovine � caprine
Genetic engineering of animals and plants has played apivotal role in research and has been directly responsible for
many significant advances in agriculture and medicine. In aneffort to determine the biological function of mammalian genes,embryonic stem (ES) cells can be genetically modified viahomologous recombination to ‘‘knockout’’ single genes or spe-cific chromosomal loci, and these modified cells can be used inthe production of germ-line chimeras, which can be crossed tocreate null mutants (1–4). To date, the overwhelming majorityof research involving the creation of genetically modified mam-mals has involved work with mice. Attempts to produce knock-out animals in other mammalian species have been limited inlarge part because of an inability to derive and stably culture EScells. Some success has been obtained by producing transgenicfetal fibroblasts via homologous recombination and then byusing these cells for cloning via somatic cell nuclear transfer;however, this approach has proven to be extremely inefficient,time consuming, and costly (5–7).
RNA interference (RNAi) is a conserved biological responseto double-stranded RNA, which mediates resistance to bothendogenous parasitic and exogenous pathogenic nucleic acids, aswell as regulates the expression of protein-coding genes (8).Recent advances involving the use of RNAi-based technologiespromise alternative approaches for the stable silencing of genesin a variety of different animal species including mammals.Expression constructs producing 19–29 nucleotide-inverted re-peats that form short double-stranded RNA hairpins or shorthairpin RNAs (shRNAs) have been demonstrated to be effectivein eliciting gene silencing (9–12). This method of producing
interfering RNAs enables induction of silencing by ‘‘classical’’DNA expression vectors and has thus become adaptable to cellculture and the production of transgenic animals, as well asintroduction into adult mammals by using established genetherapy vehicles (11). Several examples of this approach havealready been reported, including recent data from our laboratory(13), demonstrating that gene constructs expressing shRNAstargeting specific genes can be stably incorporated into thegenome of mouse ES cells and used to create transgenic miceexhibiting a phenotype analogous to that of the knockout animal.Importantly, this RNAi-based suppression was passed throughthe germ line as a dominant trait.
In more recent work, several studies involving mice have nowdemonstrated that lentiviral vectors can be used to deliverexpression constructs encoding shRNAs into early stage em-bryos to produce transgenic mice in which individual genes havebeen targeted for silencing (14–16). Because these new meth-odologies neither rely on homologous recombination nor aredependent on deriving ES cells, it should be feasible to adapt thistechnology to the production of transgenic animals in speciesother than mice, including livestock.
With the recent concerns over prion-mediated diseases (trans-missible spongiform encephalopathies) in livestock and theirpotential transmission to humans, we decided to test an RNAi-based technique for silencing the expression of the prion protein(PrP) in goats and cattle. Studies have shown that reduction ofPrP expression is, in itself, sufficient to prevent infection onexposure to the pathogenic conformer of PrP. In one geneticbackground in mouse, loss of PrP expression gave completelynormal mice with no obvious phenotype (17). In another back-ground, PrP loss was associated with disruption of sleep cycles,but it is unclear the degree to which such a phenotype wouldmanifest itself in other out-bred strains of mice, let alone in otherspecies (18). Therefore, suppression of PrP by genetic engineer-ing presents a reasonable approach for producing disease-resistant livestock and, as such, preventing transmission of priondiseases from animals to humans. In this article, we describe astrategy for using RNAi-based techniques to create a clonedtransgenic goat fetus with dramatically reduced expression ofPrP and present evidence that these techniques will also beadaptable to cattle. The approaches presented herein are alsosuitable for the creation of other types of genetically engineeredanimals that resist viral diseases or those that have improvedagricultural traits.
ResultsshRNA Design and Screening. Although the majority of recentinterest in prion disease has centered on studies in cattle, thelower biosecurity requirements for studies of scrapie prompted
Conflict of interest statement: No conflicts declared.
us to first test the possibility of using RNAi to suppress PrPexpression in another commercially important livestock animal,the goat. To identify effective targeting sequences for caprineand bovine PrP, the coding sequence was processed through acomputer algorithm that predicted a total of 24 shRNAs de-signed against the PrP mRNA (see Materials and Methods).Individual shRNAs were inserted into a lentiviral expressionvector in which the interfering RNAs were driven by the mouseH1 RNase P promoter followed by ubiquitin C promoter-drivenGFP (Fig. 1A). Such a strategy has been used to create transgenicmouse lines in which RNAi was used to stably suppress targetgene expression (10, 13).
To facilitate screening of a relatively large number of candi-date shRNAs for PrP suppression, the coding sequence for thecaprine PrP was cloned downstream of the luciferase codingregion in pGL3. The resulting expression vector would producean mRNA containing the coding sequence for Firefly luciferasefollowed by a nontranslated sequence of the PrP mRNA. Eachindividual shRNA expression vector was transfected in combi-nation with the Firefly luciferase-PrP expression plasmid and anontargeted reporter plasmid, encoding Renilla luciferase, asa means of normalization. As a control, an shRNA targeting anonrelevant sequence was transfected along with the reporterplasmids. For our initial screens, we compared the performanceof these plasmids in human embryonic kidney (HEK)-293T cellsusing transient transfection. This screen was designed to rapidlyselect those shRNAs that are efficiently processed by the RNAimachinery and loaded into the RNAi-induced silencing complex(8). In accord with previous reports, such constructs allowed usto indirectly determine the relative capacity of a given shRNA tosuppress PrP by monitoring luciferase activity (19). From thesescreens, three candidate shRNAs [F3 with 65.9% suppression
compared with the control (�0.8%), F6 (56.3 � 0.7%), and F12(86.5 � 0.7%)] were selected on the basis of their range of abilityto suppress the luciferase reporter and used in subsequentstudies (Fig. 1B).
Creation of Transgenic Cells for Somatic Cell Nuclear Transfer. In ourinitial in vivo trials, a characterized adult goat fibroblast cell linepreviously used in nuclear transfer (NT) experiments was in-fected with recombinant lentivirus carrying the construct en-coding GFP and the PrP shRNA. Whereas a fetal fibroblast cellline may have been more optimal for cell culture, the lentiviralapproach precluded any long-term culture needs. Further, theadvantage of using a characterized cell line with proven abilityto produce live offspring prompted us to proceed using the adultcell line. After infection, �30% of the cells contained the stablyintegrated transgene as evidenced by GFP expression (Fig. 2 Aand B). These cells were selected and used for somatic cellnuclear transfer to produce cloned transgenic goat embryos,which were subsequently cultured in vitro to various stages ofpreimplantation development. In many cases, GFP expressioncould be visualized immediately after the nuclear transfer pro-cedure but gradually disappeared through early embryonicdivisions only to reappear at the eight-cell stage in concurrencewith embryonic genome activation. Exemplary GFP-positivehatching blastocyst can be seen in Fig. 2 C and D. Because oneof our goals was to determine the developmental competence ofthe cloned transgenic embryos, a subset was placed in cultureovernight and transferred into synchronized recipient femalesthe following day. Each doe received 12–17 one-cell stageembryos via surgical oviductal transfer. Overall, we transferreda total of 158 presumptive cloned embryos into eight recipientsand obtained one pregnancy. The normal pregnancy rate for in
Fig. 1. Design and screening of the lentiviral shRNA expression vector. (A) Graphic representation of the lentiviral shRNA expression system used. This vectoris a modification of the plasmid described by Lois et al. (23) with insertion of the mouse H1 RNase P promoter driving expression of an shRNA. The sequence shownhere targets the caprine PrP mRNA (accession no. AY723292). (B) Percent suppression of the luciferase-PrP reporter by shRNAs targeting the PrP mRNA sequence.All data are presented as the percent reduction in luciferase activity compared with the control nonrelevant shRNA. Experiments are an average of threeindependent experiments, and actual percentages and standard deviations for the shRNAs are as follows: E2, 63.7 � 0.7%; E6, 58.5 � 0.8%; E7, 2.1 � 1.2%; E8,8.2 � 0.7%; E9, 6.6 � 1.1%; F2, 48.7 � 0.8%; F3, 65.9 � 0.8%; F6, 56.3 � 0.7%; F9, 80.3 � 0.7%; and F12, 86.5 � 0.7%.
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vivo embryo transfer is �60%, whereas NT rates are much lower,�1% survival. Thus, the pregnancy rates reported in this studyare in line with studies of goat NT (20) and with the resultspreviously recorded using this cell line (unpublished data).
Evaluation of PrP Knockdown in a Cloned Transgenic Fetus. Toexamine the capacity of the shRNA to silence gene expression ina disease-relevant manner in vivo, knockdown of the PrP had tobe evaluated in the brain. Given the need to collect brain tissueand the fact that we only had one viable pregnancy, a decisionwas made to remove the fetus to determine whether it wastransgenic and to compare PrP expression with a normal age-matched control derived by natural mating. The fetus wassurgically recovered at 81 days of gestation, and tissues wereharvested for analysis. A control goat fetus of similar gestationalage was also harvested for comparison. All tissues derived fromthe cloned fetus displayed strong GFP fluorescence consistentwith the presence of the transgene (Fig. 3). To confirm that theshRNA expression cassette was present in the genome, DNA wasisolated from brain tissue and used as template in PCR ampli-fying the region between the H1 and ubiquitin promoters. As canbe seen in Fig. 4A, the transgenic goat genome contains theshRNA expression cassette. We next assessed the ability of theshRNA to suppress PrP expression. Protein extracts were takenfrom transgenic and WT brain tissue and analyzed by Westernblot analysis. Blots were probed by using an antibody recognizinggoat PrP, which was a generous gift from Katherine O’Rourke(U.S. Department of Agricultural–Agricultural Research Ser-vice, Burns, OR) (21). As indicated in Fig. 4B, PrP expressionwas reduced by �90% in the transgenic fetus when comparedwith the control.
Because the lentivirus used to produce the nuclear donors isreplication-deficient, it is unlikely that the transgene will bepassed from the transgenic fetus to the mother; however, thispossibility has not been rigorously examined. Large-animalmodels offer a unique opportunity in this regard because of theirplacental physiology. Ungulates such as the bovine and caprineconcentrate their fetal placental villi together into discrete foci
termed cotyledons. Fetal cotyledons interact with maternalregions called caruncles and together form the functional unitsof the placentome, where maternal-fetal nutrient exchange takesplace. To determine whether the lentiviral transgene remainedrestricted to the transgenic fetal cells, cross-sectional tissuesamples of the placentome were taken and stained for GFPexpression. As can be seen in Fig. 4 C and D, GFP expression isrestricted to the external fetal component, whereas the innermaternal tissue remains GFP-negative.
Creation of Transgenic Embryos by Direct Injection of RecombinantVirus Followed by in Vitro Fertilization and Embryo Culture. Given theeffectiveness of our viral vector for delivery of transgenes intofibroblast cells growing in culture (�30%) and because theprocess of animal cloning is so inefficient, we decided to test theeffectiveness of our vector for delivery of the transgenes directlyinto early-stage bovine embryos. This strategy, based on work byHofmann et al. (22) and others (23), relies on the delivery ofinfectious viral particles into the perivitelline space of single-cellova and subsequent infection as the embryo develops. Due to theunusually high degree of sequence conservation between caprineand bovine PrP (96%), micromanipulation was used to inject 139in vitro matured bovine ova with the same recombinant lentivirus
Fig. 2. GFP expression in transgenic goat fibroblasts and cloned goatembryos. (A) Fibroblasts are shown during preparation for somatic cell nucleartransfer. (B) Expression of the shRNA-GFP transgene in primary goat fibro-blasts after integration of the lentiviral vector. (C) Bright-field image of thehatching goat blastocyst shown in D. (D) Ubiquitin C promoter-driven GFPexpression in a hatching blastocyst produced via nuclear transfer by usingtransgenic goat fibroblasts. Goat embryos were produced by somatic cellnuclear transfer by using GFP-positive transgenic goat cells seen in A and B asnuclear donors. Note the lack of fluorescence in the nondeveloping embryos.
Fig. 3. Expression of green fluorescent protein in whole-mounted tissuesfrom a cloned transgenic fetus (C–H). Images are transmitted (A, C, E, and G)and fluorescent (B–D, F, and H) light micrographs of fresh tissue samples. (Aand B) Nontransgenic uterine myometrium from a recipient doe carrying atransgenic fetus. (C and D) Fetal intestinal mesentery. (E and F) Fetal intestinallumen. (G and H) Fetal liver.
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described above. After injection into the perivitelline space, theova were fertilized and cultured in vitro. Of these ova, 42 (30%)developed to the blastocyst stage and 32 (76%) were GFP-positive (Fig. 5).
Expression of GFP was first observed at the morula stage ofdevelopment but became much more apparent during blasto-cyst formation. All embryos expressing GFP exhibited uniformfluorescence throughout the inner cell mass and trophecto-derm, without any obvious signs of mosaic expression. Theseobservations indicate that the virally delivered transgeneincorporated into the zygotic genome during early develop-ment and that expression initiated at the maternal zygotictransition as expected. Given the nature of our gene construct,expression of GFP is indicative that the shRNA targeting PrPwas also expressed. These results strongly suggest that themethods used here are effective in the production of trans-genic goats and cattle containing an expression cassette elic-iting RNAi-based silencing.
DiscussionThe ability to genetically engineer animals has become a stan-dard laboratory tool for physiological, genetic, and biomedicalresearch. However, mice represent the vast majority of trans-genic animals produced to date. Additional animal models arealso of critical importance for medical research because mice arenot completely representative of human genetics and physiology.For example, the limited life span and small size of the mouserestricts its usefulness in studies requiring long-term evaluationof test subjects (24, 25). Thus, development of treatments andcures for human diseases are sometimes better derived fromcomparative studies involving animal models other than mice. Inthis article, we provide conclusive evidence that in a large-animalsystem, lentiviral delivery of shRNAs targeting specific gene(s)is indeed effective at reducing expression of the protein in vivo.
Furthermore, these results demonstrate that lentiviral deliveryof shRNA constructs has the capacity to stably knockdown genesof interest, thus providing an efficient route to functionalgenomic research in a large-animal model.
We acknowledge that the present study yielded only a singletransgenic fetus; however, given the inefficiencies of the nucleartransfer procedure, this result is not unusual (26). Despite thisinefficiency, the length of the study was significantly less com-pared with the time required by techniques using traditionalhomologous recombination. Experiments presented here as wellas results by Hofmann et al. (22), obtained through directinjection of recombinant virus into the perivitelline space ofbovine ova, clearly demonstrate the capacity to make viabletransgenic bovine embryos. The use of direct viral injection tocreate transgenic zygotes instead of genetically modifying a cellline and producing reconstructed (cloned) embryos dramaticallyimproves the efficiency and applicability of this technology. Thistechnique, coupled with the ever improving shRNA expressioncassettes, now enables a more functional approach to be takenwhen studying gene function in animal species in which stem celltechnology is lacking but assisted reproductive technologiescurrently exist.
RNAi holds the promise of enabling production of plants andanimals that are genetically altered to produce favorable char-acteristics. Recent years have seen a greatly increased awarenessof threats posed to human health by diseases borne in livestockpopulations. Viral pathogens, such as the avian influenza strainH5N1, can be transmitted from domestic fowl to humans withoften lethal results (27). Similarly, the detection of prion-mediated diseases in cattle have elicited the imposition of trade
Fig. 4. Characterization of PrP suppression in the transgenic goat. (A) PCRamplification of the shRNA expression cassette from plasmid DNA (control), aswell as genomic DNA isolated from WT and transgenic fetal tissue. (B) Westernblot analysis of 100 and 75 �g of protein extract taken from WT and transgenicfetal brain. A residual amount of PrP can be detected in the transgenic lane;however, it is substantially reduced when compared with WT. The blot dis-played was one of two independent replicates. (C) Immunostaining of pla-centome cross-sections with an anti-GFP antibody. GFP-positive transgeniccells can be seen surrounding GFP-negative maternal tissue, indicating thatexpression of the lentivirally delivered GFP is restricted to fetal cells. (D)Negative control placentome.
Fig. 5. Transgenic blastocysts produced by in vitro fertilization and embryoculture. (A) Bright-field image of control blastocyst (noninjected ova). (B) Thesame embryo as in A viewed using fluorescence microscopy. (C) Bright-fieldimage of a bovine blastocyst that was produced by injection of an in vitromatured bovine ovum with a recombinant lentiviral vector encoding GFP andan shRNA targeting PrP, followed by in vitro fertilization and embryo culture.(D) The same embryo as in C viewed by using fluorescence microscopy. Theexpression of GFP in the embryo depicted in D demonstrates that this embryohas incorporated the transgene encoding GFP and a shRNA targeting PrP intoits genome.
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restrictions and necessitated the destruction of large numbers ofanimals, with substantial economic impact. Although carefulmonitoring of animal health and appropriate safety precautionsare a current approach to containing such diseases, there istheoretical potential for creating genetically engineered strainsof animals with a natural resistance to numerous diseases.However, genetic methods for altering livestock have thus farbeen lacking.
Previous reports demonstrated that transfection of smallinterfering RNAs was able to reduce the level of both theendogenous and infectious PrPs in cell culture, but stablesuppression of PrP in a cell line by using these small interferingRNA sequences was not demonstrated (28, 29). In this article, wereport the use of shRNAs to generate both a stable cell line anda cloned transgenic goat fetus with drastically reduced expres-sion of PrP, the causative agent of a neurodegenerative diseasethat might be transmitted to humans. Additionally, recent workhas demonstrated RNAi to be effective in eliciting stable sup-pression of the foot-and-mouth disease virus as well as porcineendogenous retroviruses, both of significant concern to agricul-ture and studies of xenotransplantation (30–32). Whereas thesemethods demonstrate stable suppression in cell culture, thesuccessful use of this technology in creating genetically modifiedlivestock is not implicit. It is very likely that the methodsdescribed herein can be adapted to the suppression of viralsequences such as influenza, porcine endogenous retroviruses,and foot-and-mouth disease, as well as targeting genes that resultin improved characteristics for the production of fiber, meat, ormilk products.
Materials and MethodsProduction and Screening of shRNAs. The coding sequences of thebovine and caprine mRNAs were processed through a com-puter algorithm that predicted a total of 24 shRNAs designedagainst the PrP sequence. Given the unusually high degree ofsequence conservation between the bovine and caprine PrPmRNA (96%), several of the shRNAs designed perfectlymatched the mRNA from both species. Individual shRNAswere ordered as antisense oligonucleotides (Sigma Genosys)and used in PCR with a sense primer homologous to the mouseH1 RNase P promoter to produce a PCR product containingthe H1 promoter directly upstream of the shRNA. Reactionproducts were gel-purified and directionally cloned into thepENTR-D entry vector (Invitrogen) by using the recom-mended protocol. The shRNA sequences were verified in theGene Technologies Laboratory at Texas A & M University.Subsequently, shRNA were inserted into a modified lentiviralexpression vector (23) containing the Gateway acceptor cas-sette by using the Clonase reaction (Invitrogen) according tomanufacturer’s recommendations.
Screening of Candidate shRNAs for PrPs Suppression. IndividualshRNAs were screened indirectly by monitoring the ability ofeach construct to silence a luciferase reporter gene containingeither the bovine or caprine PrP mRNA. This system evaluateseach shRNA’s capacity to silence a Firefly luciferase-PrP tran-script by transiently transfecting each shRNA together with thereporter gene and measuring luciferase activity by using methodsdescribed by Yu et al. (19). Briefly, the coding sequence for thecaprine PrP mRNA (accession no. AY723292) was amplifiedfrom goat testis by using RT-PCR (Invitrogen) and cloned intothe XbaI site downstream of the Firefly luciferase-coding regionin pGL3 (Clontech). Individual shRNA expression vectors weretransfected into National Institutes of Health human embryonickidney 293 cells (American Type Culture Collection) by usingcalcium phosphate along with the Firefly-PrP expression plas-mid and a nontargeted reporter plasmid, encoding Renillaluciferase, as a means of normalization. Luciferase activity was
measured by using the Stop and Glow kit (Promega) on aluminomitor according to the standard protocol. As a control, anshRNA targeting a nonrelevant sequence was transfected alongwith the reporter plasmids. Human embryonic kidney 293 cellswere used initially for the rapid screening because of their easytransfection and consistency in cell culture. A total of 24 shRNAswere screened in six pools containing four shRNAs each. Thesepools were selected for further analysis on the basis of theirability to consistently suppress the described luciferase reporterin three replicate assays. Two pools demonstrating strong sup-pression were expanded into their individual shRNAs andscreened by using the same assay.
Production and Concentration of Viral Vectors. Lentivirus was pre-pared and concentrated by using methods described by Lois et al.(23). Briefly, viral vectors were transfected into National Insti-tutes of Health human embryonic kidney 293 cells by usingmethods described above along with plasmids encoding the deltapackaging signal and a vesicular stomatitis virus glycoproteinpseudotype. Medium was changed 24 h after transfection, andcells were cultured for an additional 48 h, after which mediumwas collected and recombinant virus was concentrated by usinga standard polyethylene glycol precipitation.
Viral Infection of Fibroblasts and Nuclear Transfer. Given the pre-vious success with producing transgenic livestock by geneticallymodifying fibroblasts and then using these for animal cloning, wefirst decided to infect goat fibroblasts by using the lentiviralshRNA construct followed by selection of transgenic cells fornuclear transfer to produce cloned embryos. Caprine fibroblastswere obtained via skin biopsy from an adult male and culturedin DMEM�F-12 with 10% FBS�0.5 mg�ml gentamycin in ahumidified atmosphere of 5% CO2�air. Cells at passage twowere transferred to six-well plates (Corning). When cellsachieved 50–60% confluency, fibroblast cells were infected bydelivery of concentrated virus directly into the culture mediumalong with a 1 � polybrene solution. Cells were spun at 1,000 �g for 1 h and cultured in viral medium overnight. The next day,medium was changed, and cells were incubated for 3 days beforeassessing the expression of GFP. Once GFP expression wasestablished (�30% of exposed cells), cells were subpassed viastandard protocol and used for nuclear transfer before passagefive.
Nuclear transfer was performed as described in refs. 26 and 33.Briefly, goat oocytes were obtained from ovaries of slaughtereddoes and cultured in vitro to undergo meiotic maturation. Matureova were enucleated, and a GFP-positive donor cell was placedin the perivitelline space. Donor cells were fused to the enucle-ated ova by using two dc electrical pulses (2.0 kV�cm). Recom-bined cells were cultured in cycloheximide for 5 h, washed, andplaced in G1.3 medium (Vitrolife, Englewood, CO) in a humid-ified atmosphere of 5% CO2�5% O2�90% N2. Cloned embryoswere either transferred to the oviduct of synchronized recipientdoes on day 1 of culture or maintained in G1.3 for 3 days beforetransfer to G2.3 (Vitrolife), cultured to the blastocyst stage, andevaluated for GFP expression.
Lentiviral-Mediated Delivery of Transgenes into Bovine Zygotes. Oursecond approach to deliver the shRNA constructs into embryoswas based on a report by Hofmann et al. (22). Here, injection ofrecombinant lentivirus into bovine ova followed by in vitrofertilization and embryo culture resulted in a high proportion oftransgenic embryos. Bovine ova were obtained from a localabattoir and matured in vitro. Micromanipulation was used toinject concentrated virus into the perivitelline space of matureova. After injection, the ova were fertilized in vitro by usingstandard procedures and then cultured in vitro by using G1�G2embryo culture medium (20, 21) at 38.5°C in an atmosphere of
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5% CO2�5% O2�90% N2. After 7 days, the embryos wereremoved from the culture. The percentage of embryos devel-oping to the blastocyst stage were recorded, and the embryoswere visualized under a fluorescent microscope to determinewhether they were transgenic as indicated by the expression ofGFP.
We acknowledge the efforts of Dr. Taeyoung Shin (Texas A & MUniversity) and Suzanne Menges, Kim Green, and Drs. Bill and Gab-
riella Foxworth (Global Genetics and Biologicals, Bryan, TX) for theircontributions for the production and transfer of cloned goat embryos; wealso thank Katie Dunlap for help with placentome analysis. This workwas supported in part by Center for Environmental and Rural Health atTexas A & M University Grant P30-ES09106; U.S. Department ofAgriculture–Cooperative State Research, Education, and Extension andNational Institutes of Health–National Center for Research ResorcesGrant 1R21RR02078501A1 Service Grant 2004-35205-14192 (toM.E.W.); and the National Institutes of Health (G.J.H). G.J.H. is anInvestigator of the Howard Hughes Medical Institute.
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